OBJECTIVE: To elucidate the basic mechanisms involved in the regulation of erythropoiesis and to apply this knowledge to human erythropoietic diseases. The techniques of marrow cell culture are being used to study the effect of erythropoietin (Ep) and endotroxin on marrow erythroid precursor cells. Endotoxin has been found to greatly increase the number of day-3 BFU-E in the spleen while Ep produces a shift of these cells to the CFU-E in the spleen. We are now studying the effect of BCNU on the numbers, cell cycle, velocity sedimentation and Ep sensitivity of murine day-8 BFU-E. Preliminary results indicate that BCNU alters the velocity sedimentation and cell-cycling characteristics of day-8 BFU-E. These experiments should clarify the developmental characteristics and capacities of erythroid precursor cells by elucidating their response to Ep under different circumstances. Polycythemia produced by the Friend virus is also being studied. We have shown that Friend virus produces erythroid bursts when added to mouse marrow cells in vitro. These studies indicate that the in vitro target cells for Friend virus-induced erythroid bursts is a relatively mature erythroid precursor cell. Further studies are in progress to determine whether this cell is a subset of CFU-E or represents the day-3-5 BFU-E. These studies are being performed by velocity sedimentation and density gradient separation of mouse spleen cells from animals treated with endotoxin to enrich the day-3-5 BFU-E in their spleens. Kinetic studies have indicated that only one type of virus particle is necessary for the erythroid transformation in vitro. This particle is the spleen focus-forming virus (SFFV). It appears that MuLV-F is not required for erythroid transformation but only SFFV replication. Studies are now underway to purify the SFFV by source gradient centrifugation and affinity chromotography and to demonstrate directly that erythroid bursts can be produced without the presence of the replicating virus. The Friend virus transformation of erythroid bursts is greatly potentiated by exquisitely small amounts of erythropoietin. The mechanism of this potentiation will be studied in the forthcoming year.